car-t cell therapy · acute lymphoblastic leukemia zeller jl, et al. jama. 2007;297:1278. • the...

CAR-T cell therapy

Usanarat Anurathapan, MD

Department of Pediatrics

Faculty of Medicine Ramathibodi Hospital

Outline

• Related basic immunology

• Cellular immunotherapy

• CAR-T cell therapy

Basic immunology

• T-cell receptor

• T-cell activation and costimulation

• Effector T cells and cytotoxic T cells

T-cell receptor

• Self-MHC restriction

– Specific to a combination of an antigen and a major

histocompatibility complex (MHC), also known as a

human leukocyte antigen (HLA)

αβ and γδ T-cell receptors

Heterodimer composed of either α and β chains or

γ and δ chains

Comparison of αβ and γδ T cells

Feature αβ T cells γδ T cells

Proportion of CD3+ cells 90-99% 1-10%

TCR V gene germline repertoire Large Small

CD4/CD8 phenotype

CD4+

CD8+

CD4+ CD8+

CD4- CD8-

60%

30%

< 1%

< 1%

< 1%

30%

< 1%

60%

MHC restriction CD4+ : HLA class II

CD8+ : HLA class I No MHC restriction

Ligands MHC + peptide

antigen

Phospholipid, intact

protein

TCR multigene families

Gene Chromosome

location

Number of gene segments

V D J C

α chain 14 54 - 61 1

β chain 7 67 2 14 2

γ chain 14 14 - 5 2

δ chain 7 3 3 3 1

TCR rearrangement

TCR complex: TCR-CD3

Associated on its membrane with CD3,

multicomponent signal-transducing complex

T-cell activation & costimulation

T-cell activation

• The central event in the generation of both

humeral & cell-mediated immune response

• Signal 1 = Initiated by interaction of TCR-

CD3 complex with a processed antigenic

peptide bound to either HLA class I

(CD8+ cells) or class II (CD4+ cells) on

the surface of an antigen-presenting cell

TCR & CD4 or CD8 coreceptor

Affinity of TCR for peptide-MHC complexes is enhanced

by CD4 or CD8 coreceptors

TCR-CD3 mediating signaling

T-cell costimulation

Signal 2 = costimulatory signals are required for

full T-cell activation

T-cell activation & differentiation

Effector T cells

CTLs kill cells in two ways

Necrosis vs Apoptosis

Cytotoxic T cells

Cancer immunotherapy

• Monoclonal antibodies: -mab

• Cytokines & T cell subsets

• T cell activation: CTLA-4 Ig, anti-CD3

• B-cell based therapy: anti-CD20

• Cellular therapeutics

Cellular immunotherapy

• Active immunization

– Vaccines: DNA, protein, whole-cell, dendritic cell

• Adoptive immunotherapy

Adoptive immunotherapy:

T cells to induce tumor regression

1

Blood draw

Adapted from Ronald Levy, MD

*Antigen

Specificity*

2

T-cell product

generation

3

Infusion 4

T cells

Adoptive immunotherapy

in allogeneic setting

1

Blood draw

Adapted from Ronald Levy, MD

*Antigen

Specificity*

2

T-cell product

generation

3

Infusion 4

T cells Donor

Recipient

Types of adoptive T cell therapy

• Natural tumor-specific T cells

– Natural-killer cell (NK cell)

– Tumor infiltrated lymphocyte (TIL)

– Cytotoxic T lymphocyte (CTL)

• Genetic-modified tumor-specific T cells

NK cell

TIL

• Solid tumor

– Melanoma

– Pancreatic cancer

– Prostate cancer

CTL specifically kill tumor cells

HLA-restricted cytotoxicity effect

Types of adoptive T cell therapy

• Natural tumor-specific T cells

• Genetic-modified tumor-specific T cells

Principles of gene & cell therapy for cancer

• To exert cytotoxicity on cells in neoplastic

tissue

• To stimulate anti-tumor immune responses

• To interfere with cellular signaling pathways

conferring malignant properties

Singh, HM et al. Expert Opin Biol Ther 2014:15;1-12.

Modalities of gene & cell therapy for

cancer Modality Gene delivery systems Transduced cell types

Gene addition or editing

(in vivo or ex vivo gene therapy)

Lentivirus

Gammaretrovirus

Adenovirus Adeno-associated virus

Tumor cell

Stromal cell Immune cell

Genetically engineered cell

therapy (ex vivo gene transfer)

Transposon system

DNA/RNA transfection Nanoparticle

T lymphocyte

Antigen-presenting cell

Isolated tumor cell

(whole cell vaccine)

Mesenchymal stem cell Bacterial cell

Oncolytic virotherapy

(infection +/- in vivo gene transfer)

Selectively replicating

virus, unmodified or recombinant

Target tumor cell Cellular delivery vehicle

Singh, HM et al. Expert Opin Biol Ther 2014:15;1-12.

How does it work?

http://stemcells.nih.gov/StaticResources/info/scireport/images/4_3.jpg

Types of adoptive T cell therapy

• Natural tumor-specific T cells

• Genetic-modified tumor-specific T cells

– Transgenic TCR-modified T lymphocyte

– Chimeric antigen receptor-modified T lymphocyte

Transgenic TCR T cells

Chhabra A. The Scientific world J. 2011; 11: 121-9

What are the disadvantages?

Chhabra A. The Scientific world J. 2011; 11: 121-9

Genetic modification that

enhanced T cell targeting

Anurathapan U, et al. Cytotherapy. 2013; Epub ahead of print

What is CAR?

Chimeric Antigen Receptor

What is CAR?

Chimeric Antigen Receptor

What is CAR?

Chimeric Antigen Receptor

How can CAR work?

T cell

CAR

Anurathapan U, et al. Cytotherapy. 2013; Epub ahead of print

CAR detect specific-TAA on tumor cells

Tumor-associated antigen (TAA)

Tumor cell

T cell

CAR

Anurathapan U, et al. Cytotherapy. 2013; Epub ahead of print

Dimerization of CAR produce signal

T cell

ZAP70

Signal transduction

CD3ζ

membrane

Cytotoxicity

Genetically modified T cells redirect the

immune response against cancer cells

VL

CL

VH CH

CH2

CH3

Tumor specific

Antibody

CH2

CH3

TM

TAA

scFv VH

VL

T cell

Signaling

domains

Clone into

Expression vector

Gene transfer

Into T cells

Cancer cells

(TAA+)

CAR-modified T cell

Ex vivo expansion

of modified T cells

Cytotoxicity

Membrane

Evolution of CAR

First

Exodomain

Hinge

Endodomain

Second Third Generation

Costimulatory molecules

CD28, 41BB, OX40

Signaling domain

CD3ζ

Survival

Proliferation/cytokine production

Fourth

Chimeric cytokine

receptor

IL4/IL2, IL4/IL7

CAR T cells for clinical application

• Be produced and expanded according to GMP

guidelines and in larger scale

• GMP-graded materials and reagents will be used

during all processes

• All cell preparation will also be conducted in GMP

facilities

• Be tested for viability, expression, efficacy and

sterility prior to administration

Acute lymphoblastic leukemia

Zeller JL, et al. JAMA. 2007;297:1278.

• The most common

cancer in children

• Fever, bleeding,

pallor, bone pain

• Chemotherapy for

3 years

CAR-T against CD19+ leukemic cells

Bridget M, et al. JAMA. 2017;318:2167.

Phase I clinical trials: meta-analysis

• 133 pts with B-cell malignancies

• 119 pts with eligible for response rate evaluation Zhang T, et al. Oncotarget. 2015. 33961.

14 Clinical trials

Zhang T, et al. Oncotarget. 2015. 33961.

Forest plot for overall response rates

Zhang T, et al. Oncotarget. 2015. 33961.

Better response rate in ALL: 93 (65-100)%

ALL

CLL

Lymphoma

Overall

Zhang T, et al. Oncotarget. 2015. 33961.

Better response rate with lymphodepletion: 88 (65-100)%

Lymphodepletion

No lymphodepletion

Zhang T, et al. Oncotarget. 2015. 33961.

Better progression-free survival with

>108 infused CAR T cells

Zhang T, et al. Oncotarget. 2015. 33961.

Conclusion

• High clinical response rate of CAR-CD19 T

cell-based immunotherapy

• Lymphodepletion & increasing no. of infused

CAR-CD19 T cell have positive correlation

with the clinical efficacy

Zhang T, et al. Oncotarget. 2015. 33961.

CAR T cells for solid tumors

• Currently, around 100 CAR T cell trials

registered at the ClinicalTrial.gov

• Most of studied targets

– EGFRvIII for glioblastoma

– GD2 for neuroblastoma

– Mesothelin for various epithelial cancers

Major obstacles to the efficacy

Selected CAR T cell trials

Antigen Indication Lymphodepletion Route of administration

CD70 Pancreatic/Renal Cell/Breast/ Ovarian CY/FLU Systemic

CD171 Neuroblastoma N/S Systemic

EGFRvIII Recurrent Glioblastoma/-sarcoma - Intracerebral

ErbB Head and Neck Cancer - Intratumoral

FAP Malignant Pleural mesothelioma - Intrapleural

GD2 Neuroblastoma CY/FLU Systemic

GPC3 Hepatocellular Carcinoma Cy/FLU Systemic

HER2 (ErbB2) Glioblastoma - Intracerebral

IL13Rα2 Glioblastoma, Brain Tumors - Intracerebral

MET Melanoma, Breast Cancer - Systemic

Mesothelin Breast/Cervical/Pancreatic/Ovarian/Lung Cancer CY/FLU Systemic

MUC-16 Ovarian/Fallopian tube Carcinoma CY/FLU Systemic

PSCA Prostate/Pancreatic Cancer - Systemic

ROR1 Triple Negative Breast Cancer/NSCLC CY/FLU Systemic

Combinatorial antigen targeting for

solid tumors

Somsak Prasongtanakij, Amornrat Tungprasitipap,

Maytawan Thanunchai, Thitinee Vanichapol,

Jiraporn Jirakkakul, Korakot Atjanasuppat,

Sassawat Lertrit-anan, Bunyada jittorntrum,

Vichaya Suttisunhakul, Suradej Hongeng,

Usanarat Anurathapan

Faculty of Medicine Ramathibodi Hospital, Mahidol University

Development of cellular therapy

for the treatment of cancers

Phase1/2 clinical trial

• Relapsed/refractory B cell leukemia patients

• Age 1-18 y/o

• No co-existing diseases

• Available donors for stem cell transplantation

• Priming with 1.5-3g/m2 of cyclophosphamide

• Start with 5x105 cell/kg in the first 3 patients

Protocol flow chart

Maximum tolerated dose

total T cells (cells/kg)

Tier 1 5 x 105

Tier 2 1 x 106

Tier 3 2 x 106

Tier 4 4 x 106

Enrolled subjects Pts with

complications Plan

Upto 3 pts 0 Increase CAR-CD19 T cell dosage to the next tier

upto 3 pts 1 Enrolled 3 more pts receive CAR-CD19 T cell

dosage at the same tier

4 – 6 pts 1 Increase CAR-CD19 T cell dosage to the next tier

4 – 6 pts 2

Assume the CAR-CD19 T cell dose is jmaximum

tolerated dose, the next enrolled 3 pts receive the

previous CAR-CD19 T cell dose to assure that is no

complications

CAR-CD19 T cells release criteria

Test Specimen Specification RESULT

Viability by trypan blue Cell product >70% viable 97.30% (Total live cell = 252-295 x 106)

Phenotyping Cell product >10% CAR-CD19 T cells 16-21%

Potency Cells on day 7

of culture

> 20% lysis at 20:1 of effector to target ratio

32.92-38.97%

Mycoplasma test Final product Negative Negative

Presence of bacteria by light microscope

Final product Negative Negative

Sterility Bactec Final product Negative Negative

N=2

GD2-expressing neuroblastoma

• Disialoganglioside antigen that is expressed on

neuroblastoma cells

• Patients with neuroblastoma were found to have

significantly elevated free GD2 levels in serum

• In normal tissues, GD2 expression is largely

limited to neurons, skin, melanocytes, &

peripheral pain fibers suitable for targeted

antitumor therapy

CAR-GD2 (1st Gen) CAR-GD2 (2nd Gen)

GD2-binding domain

“A kindly gift from Baylor College of Medicine”

CAR-GD2 second generation

62

0

10

20

30

40

50

60

70

80

90

100

CD3 CD4 CD8 CD25 CD27 CD62L CD45RA CD45RO

Day0

Day4

The effector cell numbers increase after CD3/CD28 activation

Immunophenotyping

63

CAR expression

• Using qRT-PCR

• Extract total RNA from CAR T cells

PCR product

CAR mRNA expression by RQ-PCR

100 bp

200 bp

300 bp

Ct value Normalized ct

CAR GAPDH CAR/GAPDH

Mock - 16.27 -

GD2 Full 24.43 15.97 1.53

GD2 Int 24.5 16.1 1.52

GD2 Short 24.05 16.31 1.47

Mock Full Int Short

Product size 281 bp

•Effector: Mock or CAR-GD2 T cells

•Target: SH-SY5Y (GD2 positive NB), SK-N-SH

(GD2 negative NB)

•Effector : Target ratio 20:1

•Labeling effector cells with 0.25 µM CFSE (FITC)

•4 hours co-cultures

•All cells were stained with 7-AAD

Cytotoxicity test using flow cytometry

GD2 expression of NB cell lines

18.76%

96.15%

GD2 positive

SH-SY5Y cell line

GD2 Negative

SK-N-SH cell line

0

10

20

30

40

50

60

70

80

90

100

SH-SY5Y SK-N-SH HEK

% s

pe

cifi

c ly

sis

Mock

Full

Int

Short

SH-SY5Y: GD2 positive NB cell line SK-N-SH: GD2 negative NB cell line HEK: human embryonic kidney cell line

Cytotoxicity test: 4h

Effector to target ratio 20:1

Questions?